A characteristic feature of chronological aging is cline of organ function. Manifestations of this decline in human skin include fragility, impaired wound healing, increased incidence of cancer, reduced elasticity and tone, uneven color, and increased roughness. All of these features are attributable, at least in part, to impairment of the structural integrity of skin connective tissue. The structural component of skin connective tissue is the dermal extracellular matrix (ECM), which is primarily composed of fibrillar type I collagen framework. The structure and function of this type I collagen framework is modulated through direct and indirect interactions with hundreds of additional proteins, which, like type I collagen, are primarily synthesized and secreted by dermal fibroblasts. Fibroblasts adhere to the ECM through integrins, which are a family of specific cell surface ECM receptors. ECM-fibroblast interactions inform and control both ECM and fibroblast functions via integrin attachment sites. These sites regulate function through two interrelated mechanisms: 1) conventional signal transduction, mediated by kinase cascades, and 2) mechanical force transduction, mediated by cytoskeletal machinery and intracellular scaffold proteins. During the aging process, fibrillar type I collagen is fragmented. This fragmentation deleteriously impacts skin health by directly compromising mechanical stability and by destroying ECM sites for fibroblast attachment. Our data indicate that these two factors conspire to reduce mechanical tension within fibroblasts in human skin in vivo. Reduction of mechanical tension manifests as change of fibroblast shape from stretched to collapsed. Fibroblast collapse is significant, because a fundamental property of cells is the connection between shape and function. Based on our preliminary data, we hypothesize that fragmented ECM accumulates with aging and causes fibroblasts to collapse, resulting in reduced ability to respond to transforming growth factor-beta (TGF-2), which is the primary driving force for ECM production. Reduced collagen production further deteriorates ECM structural integrity, leading to further fibroblast collapse, further resistance to TGF-2, and further diminishment of collagen production. This self-perpetuating decline of ECM/fibroblast functions eventuates in thin, fragile skin, characteristically seen in the elderly. To test this hypothesis, Specific Aim 1 will investigate molecular mechanisms by which the fragmented ECM microenvironment regulates TGF-2-dependent collagen production. Specific Aim 2 will investigate the impact of increasing mechanical tension in the dermis of aged human skin, by injection of space filling material, on fibroblast shape and function. Specific Aim 3 will delineate mechanisms by which fragmented collagen matrix down-regulates type II TGF-2 receptor. Specific Aim 4 will investigate mechanisms by which type I collagen-binding integrins sense, signal, and regulate fibroblast shape/mechanical tension in response to fragmented ECM microenvironment. The proposed studies are significant because they will: 1) advance knowledge of molecular mechanisms that are responsible for age-dependent decline of human skin function, 2) provide a new paradigm for understanding the impact of age-dependent ECM alterations on tissue function, and 3) enable development of new modalities to improve the health of the elderly. The proposed studies are novel because they depart from the cellular- centric view of aging to address the role of dynamic interplay between cells and their ECM microenvironment during the aging process. PUBLIC HEALTH RELEVANCE: The primary objective of this proposal is to investigate molecular mechanisms that are responsible for reduction of type I collagen synthesis in chronologically aged human skin. Loss of type I collagen deleteriously alters the structural integrity of skin connective tissue, and thereby impairing skin function and promoting age-related skin diseases. Our findings from the initial grant demonstrate that age-related reduction of type I collagen is mediated by impairment of the TGF-2 pathway in dermal fibroblasts. This proposal builds on our previous findings and will test the hypothesis that accumulation of aged-related alterations of the structural/mechanical/functional properties of the collagenous extracellular matrix in human dermis impairs dermal fibroblast functions, including TGF-2-dependent collagen production. This hypothesis is novel because it focuses on the importance of the extracellular matrix, rather than inherent cellular deficits, as a driving force for the aging process in humans.